DR.RAGHAVENDRA.H.GOBBUR
PROFESSOR OF PEDIATRICS
B.L.D.E.UNIVERSITY’S Shri.B.M.Patil MEDICAL COLLEGE ,BIJAPUR
rhgobbur@gmail.com
Newer modalities in TB diagnosis
Guest lecture given at state PEDICON 2011
DR.RAGHAVENDRA.H.GOBBUR
PROFESSOR OF PEDIATRICS
B.L.D.E.UNIVERSITY’S
Shri.B.M.Patil MEDICAL COLLEGE ,BIJAPUR.
rhgobbur@gmail.com
Newer modalities in TB diagnosis
•
•
•
•
•
•
IGRA assay Interferon (IFN)-γ Assay
Microscopy LED
Culture :Liquid Medias: BACTEC,
BAC T/ALERT 3D, MGIT
DNA NAAT: Real-time PCR, LIPA
Enzyme Assay: ADA
QUANTIFERON-TB GOLD
in place of Mantoux test .
INTERFERON G ASSAY (IGRA)
IFN-γ
QUANTIFERON-TB GOLD .
IN VITRO TEST
ESAT-6 ,CFP-10 synthetic peptides are used
(Absent in BCG and most NTM)
stimulate T-cells from infected people
releasing IFN-γ, from These T-cells
*early secretory antigenic target-6
**culture filtrate protein-10
LTBI
-
TST
V/S
V/S
DISEASE ?
IGRA
QUANTIFERON-TB GOLD .
• Objective , and controlled test
Reduces subjectivity in TB diagnosis
• Simple diagnostic cut-off (> .35 IU/ml IFN-γ = + )
Straight forward positive/negative interpretation
• Eliminates 2 step testing
• No ‘booster’ effects in-vitro
• Faster turn-around, results in 24 - 48 hours
• Results are electronic (computer generated reports)
INDIAN STUDY USING QUANTIFERONTB GOLD
•
Dogra S, Narang P, Mendiratta DK, Chaturvedi P, Reingold AL, Colford JM Jr, Riley LW, Pai M.
Comparison of a
• whole blood interferon-gamma assay with tuberculin skin testing
• .
( J Infect 2007; 54:267–76.)
•
Compared QFT to the TST in 105 children ( suspected of TB, or had contact with an index
case).
•
•
•
•
•
11 children (10.5%) were QFT positive, whereas the
TST was positive in 15 (15%) at ≥5mm, 11 (10.5%) at ≥10mm, or 4 (4%) at
≥15mm
.
Concordance of TST with QFT was high (95%) at the 10mm
TST cut off
• All subjects with≥15mm TST , were QFT positive.
• There were
•
no indeterminate QFT results, despite 40% children being <4 years old
,
and 57% of them being malnourished.
SUMMARY of IGRA TESTING
IGRAs are recommended for
1. Contacts of active TB
Close contacts (HIGH RISK) TST OR IGRA
if either is positive, treat for “L TB I” (latent infection)
Casual contacts (LOW RISK) can have IGRA confirmation
if TST is positive to verify infection v/s BCG or MOTT
2. Immune compromised “suspected child"
TST first, if negative do IGRA and
if IGRA positive treat as LTBI
M.TB. STAINING BY ZEIL-NEILSON STAIN
• >1,000 Organism per ml sputum required for
ordinary microscope.
• Fluorescent, LED microscope detects even 100
M.TB. organism per ml
AFB + SPUTAM SMEAR
fluorescent LED
microscope based on the proven Primo
Star platform.( FIND/Zeiss microscope offers superior optics,
FIND and Carl Zeiss
reflected light illumination, easy switch from bright field to fluorescent
light)
MYCOBACTERIAL CULTURE
Culture remains the gold standard for lab confirmation of TB
Advantages:






Increases number of case detection
Detects cases among smear negative patients
Establishes viability of organisms
Distinguishing between Mycobacterial species
Helps in performing DST (drug sensitivity test)
Helps in diagnosing cases of treatment failure
Limitations:




Expensive
Require enriched media
Require considerable expertise
Time consuming
Processing of sputum with CPC Method
If delay of more than 48 hours between collection
and processing is anticipated, the sputum should be
collected with 1%CPC and 2%NaCl2
CPC acts as homogenizing and decontaminating
agent
It helps in retaining viability of Tubercle bacilli up to
7 days
These specimens should not be treated with NaOH
( Petroff’s)
Culture: Extra-Pulmonary Samples
Aseptically collected samples
Body fluids:
Spinal ,Pleural, Pericardial, Synovial, ascitic, Blood, Pus & Bone
marrow
Tissues:
Lymph node, Needle biopsies or Tissue biopsies
Specimens known to contain contaminating flora:
Gastric lavage, Bronchial washings & Urine
L J MEDIA
CORD LIKE GROWTH OF M.TB. IN MEDIA
NEWER CULTURE METHODS for M.TB.
• Microscopic Observation of Broth Culture &
• MODS
Micro Colony Detection System (slide culture)
• Septi-check AFB : Non radiometric, Non automated
• MGIT 960
: Automated. monitors every 60min.
O2 utilization, Intensification of O2 quenching fluorescent
dye
• MB/BAC T - ALERT : Non radiometric, colorimetric
detection of CO2
•
BACTEC Radiometric
BACTEC 460 TB System(radio metric)
 Developed in 1969 by Deland and Wagner.
Principle
 BACTEC 12B vial , utilize 14C labeled substrate (fatty acid)
 On inoculation, mycobacteria, grow & release 14CO2.
 The BACTEC instrument measures quantitatively the radioactivity
on a scale ranging from 0-999, as GI.(Growth Indicator)
 The daily increase in GI is proportional to growth in the medium.

DST
Drug Susceptibility Test

When ATT is introduced in the medium

reduced production of 14CO2 and decrease in GI.
MB BACT-ALERT 3D
LIGHT EMITTING SENSORS
The MGIT 960 System
The MGIT 960 system is a non-radiometric
automated system that uses the MGIT media &
sensors to detect the fluorescence.
Advantages:
-The system holds 960 plastic tubes which are
continuously monitored.
- Early detection with the machine monitoring &
reading the tubes every hour.
II Mycobacteria Growth Indicator Tube
(MGIT)
Tube contains modified Middle brook 7H9 broth
base with OADC enrichment & PANTA
antibiotic mixture.
All types of clinical specimens, pulmonary as
well as extra-pulmonary ( except blood ) could
be cultured on this type of media.
The OADC supplement
O ----- Oleic acid ( Metabolic stimulant)
A ----- Albumin ( to bind toxic free fatty acid )
D ---- Dextrose (Energy source )
C ----- Catalase ( Destroy toxic peroxides that
may be present in the medium )
The PANTA antibiotic mixture
P ---- Polymyxin B
A ---- Amphotericin B
N ---- Nalidixic acid
T ---- Trimethoprim
A ---- Azlocillin
+/- Vancomycin
The antibiotic mixture inhibits the growth of
contaminating bacteria.
Principle of the procedure:(MGIT)
A fluorescent compound (which is sensitive to
O2) is embedded in silicone on the bottom of
the tube.
The actively respiring microorganisms consume
the oxygen & allow the fluorescence to be
observed using UV trans-illuminator lamp.
III Polymerase Chain Reaction (PCR) &
Gene probe
Nucleic acid Amplification Tests polymerase enzymes
amplify specific DNA sequences, using Nucleic
acid probes, using DNA extracted from MTB in the
sample.
Advantages:
- Rapid procedure ( 3 – 4 hours)
- High sensitivity (1-10 bacilli / ml sputum)
CDC recommends NAAT for all suspected TB cases
PCR ASSAY
The thermal cycling,
DNA melting separates the strands of DNA double helix at 95°C
Heat-stable DNA Taq polymerase, ( Bacteria Thermus aquaticus.)
Enzymatically assembles new DNA strands(selectively amplify )
using DNA primers ( DNA oligonucleotides.) & template(each
strand) at 55 °C
The selectivity of PCR results from the use of primers that
are complementary to the DNA region targeted for amplification
under specific thermal cycling conditions.
RR
REALTIME-PCR ASSAY
• A TB specific primer and probe mix is provided and
this can be detected through the FAM channel.
• The primer and probe mix exploits the so-called
TaqMan® principle.
• During PCR amplification, forward and reverse primers
hybridize to the TB DNA/Cdna
• . A fluorogenic probe is included in the same reaction
mixture , it consists of a DNA probe labelled with a
• 5`-dye (reporter) and a 3`-quencher. (5’3’DQ)
• During PCR amplification, the probe is cleaved and the
Reporter dye and Quencher are separated.
• The resulting increase in fluorescence can be detected
on a range of real-time PCR platforms
PCR ASSAY
• The PrimerDesign™ genesig Kit for Mycobacterium
Tuberculosis (TB) Genomes is designed
• for the in vitro quantification of TB genomes.
• The kit is designed to have the broadest detection profile
possible whilst remaining specific to the TB genome.
• The primers have 100% homology with all other reference
sequences in the NCBI database.
• Fig 1 Accession numbers for detected TB isolates.
• CP000717.1, CP000611.1, AM408590.1, U43540.1,
AE000516.2, BX842583.1, BX842577.1,
• BX842572.1, BX248339.1, U35021.1, U35017.1,
AF041819.1, BX248346.1, BX248334.1,
Disadvantages:
- Very expensive.
- Require specialist training & equipment.
- False positive results.( CONTAMINATION)
- Can not differentiate between living & dead
bacilli. .
- Sputum specimens (3%--7%) might contain
inhibitors that prevent or reduce amplification
and cause false-negative NAA results.
RAPID RECOGNITION OF DRUG RESISTANCE
• PCR PROBES ARE AVAILABLE
•
•
•
•
KAT-gene
INH
RESISTANCE
RPO gene
RIFAMPICIN
RESISTANCE
GYR –A
FLUROQUINOLOE RESISTANCE
LIPA( Line Probe Assay ) amplified DNA is applied to
strips with probe for M.TB. And Rif. resistance
MODS versus other culture methods*
Method
Pos.each
Pos. by
Method(%) atleast one
cult.(%)
Sens.
%
Median
detection
days
Auramine 0
76
98
78
MODS
89
97
92
9 (4-31)
MGIT
88
95
93
10 (3-39)
LJ
73
96
76
24 ( 6-59)
Micro COL 7H11
75
96
78
14.5(4-28)
PCR
81
90
90
* Based on 172 samples
Caviedes.L. et al J..Clin.Microbiol. 2000, 38, 1203
Identification of M. tuberculosis
from the growth
Growth temperature 35o-37oC only
No pigmentation
Niacin positive
Catalase negative at 68oC
No growth on LJ medium containing PNB
Positive reaction for nitrate reduction
Differentiation of Mycobacteria
Colony
morphology
M.tuberculosis
NTM
Rough, eugonic
Mostly smooth
Growth at 37oC
+
+/ -
Growth at 25oC
-
+
Pigmentation
-
+/ -
Niacin
+
-
PNB
-
+
Nitrate reduction
+
-
Catalase at 68oC
-
+
Can high drug dosage still have an
effect on resistant strains?
Isoniazid
Mutants
katG – high
MIC
inhA – low
MIC
Quinolones Mutants Mainly in
gyrA – low
MIC
Early clinical
trial
Guinea-pig study
Evaluation of different methods of
diagnosis
As regards the time:
MGIT shortest time to positivity at 13.3 days
BACTEC 460 system 14.8 days
& for L J medium 25.6 days .
As regards the no. of culture yield:
The best yield, was with BACTEC 460, followed
by BACTEC MGIT 960 , & then with L J
medium.
As regards contamination rate:
L J medium (17%) had the highest contamination
rate (Tortoli E, Cichero P,Et al. 1999) then the
MGIT 960 ( 10.0% )
Compared with radiometeric system (3.7%)
SUMMARY
Useful newer modalities
3idiots?
• QUANTIFERON-TB GOLD
• MB BACT-ALERT 3D LIGHT EMITTING SENSORS
• PCR PROBES for antibiotic resistance
•
INH,REF,ETH,FLORO Q
Tuberculosis (TB) Diagnostic Tests in Use, Recently Endorsed by the World Health
Organization (WHO), and in Later Stages of Development.
Dorman S E Clin Infect Dis. 2010;50:S173-S177
© 2010 by the Infectious Diseases Society of America
Tuberculosis (TB) Diagnostic Tests in Use, Recently Endorsed by the World Health
Organization (WHO), and in Later Stages of Development.
• BACTEC Myco/F�Sputa Culture Medium, for
use with the BACTEC 9000MB System to
detect mycobacteria species in clinical
samples.
• BACTEC Myco/F Lytic.
•
•
•
•
•
•
•
•
•
•
•
•
•
Dogra S, Narang P, Mendiratta DK, Chaturvedi P, Reingold
AL, Colford JM Jr, Riley LW, Pai M. Comparison of a whole
blood interferon-gamma assay with tuberculin skin testing
for the detection of tuberculosis infection in hospitalized
children in rural India. J Infect 2007; 54:267–76.
An Indian study that compared QFT to the TST in 105 children who
were suspected of having TB, or had contact with an index case.
In this study 11 children (10.5%) were QFT positive, whereas the TST
was positive in 15 (15%) at ≥5mm, 11 (10.5%) at ≥10mm, or 4 (4%) at
≥15mm. Concordance of TST with QFT was high (95%) at the 10mm
TST cut-off. All ≥15mm TST subjects were QFT positive.
There were no indeterminate QFT results, despite 40% of the children
being <4 years old and 57% of them being malnourished.
SUMMARY
IGRAs are recommended for
1. Contacts of active TB
Close contacts (HIGH RISK) can get both TST and IGRA
and if either is positive, be treated for LTBI
Casual contacts (LOW RISK) can have IGRA
confirmation if TST positive to verify
infection vs BCG or MOTT
2. Immune compromised
TST first, if negative do IGRA and if IGRA positive
treat as LTBI
3. Low risk people who are TST positive
Do an IGRA, if positive consider as LTBI
FIND and Carl Zeiss Micro Imaging GmbH
have co- developed a fluorescent LED
microscope based on the proven Primo
Star platform. FIND/Zeiss microscope
offers superior optics, reflected light
illumination, easy switch from
brightfield to fluorescent light
Components of the post-research-and-development process for promising new tuberculosis
(TB) diagnostic technologies.
Dorman S E Clin Infect Dis. 2010;50:S173-S177
© 2010 by the Infectious Diseases Society of America
Reporting of culture results
Report
Reading
No Growth
•
Negative
1 – 19 colonies
•
Positive
•
Positive
(1+)
•
Positive
(2+)
•
Positive
(3+)
•
Contaminated
20-100 colonies
>100 colonies
Confluent growth
Contaminated
( No.of colonies)
INDIAN STUDY USING QUANTIFERONTB GOLD
•
Dogra S, Narang P, Mendiratta DK, Chaturvedi P, Reingold AL, Colford JM Jr, Riley LW, Pai M.
Comparison of a
• whole blood interferon-gamma assay with tuberculin skin testing
• for the detection of tuberculosis infection in hospitalized
• children in rural India. J Infect 2007; 54:267–76.
•
Compared QFT to the TST in 105 children ( suspected of TB, or had contact with an index
case).
•
•
•
•
11 children (10.5%) were QFT positive, whereas the
TST was positive in 15 (15%) at ≥5mm, 11 (10.5%) at ≥10mm, or 4 (4%) at
≥15mm.
Concordance of TST with QFT was high (95%) at the 10mm
• TST cut-off. All ≥15mm TST subjects were QFT positive.
• There were no indeterminate QFT results, despite 40% of the children
• being <4 years old and 57% of them being malnourished.
Thank you